TREATMENT WITH OPIOID ANTAGONISTS AND mTOR INHIBITORS

ABSTRACT

Embodiments of the invention provide methods of treating a disorder or disease characterized by cellular proliferation and migration by co-administering a synergistically effective amount of an mTOR inhibitor and a μ-opioid receptor antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/179,095, filed on Feb. 12, 2014, which is a continuation of U.S.application Ser. No. 12/933,784, filed on Oct. 14, 2010, now U.S. Pat.No. 8,685,995, which is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/US2009/037825, filed on Mar. 20, 2009,which claims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 61/038,577, filed on Mar. 21, 2008, the completedisclosures of which are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

INTRODUCTION

Cell growth and proliferation are normal ongoing processes in all livingorganisms, involving numerous factors and signals that are delicatelybalanced to maintain regular cell cycles. Whether or not mammalian cellswill grow and divide is determined by a variety of feedback controlmechanisms, such as the availability of space in which a cell can grow,and the secretion of specific stimulatory and inhibitory factors in theimmediate environment.

mTOR is a large polypeptide serine/threonine kinase of thephosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family. mTORlies downstream from the PI3K pathway, and functions as an intermediaryin a variety of cell signalling events to regulate cell growth andproliferation. mTOR activity is regulated by the serine/threonine kinaseAkt, and recent evidence indicates that these kinases interact through acomplex feedback inhibition pathway. mTOR modulates cell replication bycontrolling translation of key proteins that are required forprogression of the cell cycle through the G1 to the S phase. That is,mTOR controls the translation of specific mRNAs via regulation of thephosphorylation state of several proteins involved in the translation ofmRNA, mainly 4E-PB1, P7056K and eEFZ.

The mTOR pathway, with its PI3K and Akt constituents, is a criticalregulator of the proliferation of cells that responds to nutrients,hormones and growth factors, such as VEGF. Growth factors can activatePI3K signaling by binding to cognate cell surface receptors, therebyinitiating a signaling cascade through Akt that results in theactivation of mTOR. Recent studies have demonstrated that mTORinhibitors have antiproliferative and antiangiogenic effects byinhibiting both growth factor-mediated signaling and growth factortranslation.

In cancer cells, multiple dysregulation mechanisms within the PI3Kpathway upstream of mTOR have been documented as causing increased mTORactivity, and consequently, increasing tumor growth. Thus, dysregulationof the mTOR signaling pathway has been implicated in the progression ofcancer, and inhibitors of mTOR are currently being investigated ascancer therapeutic agents. mTOR inhibitors are also potentimmunosuppressive agents. One such agent, sirolimus, is currently beingused for the prophylaxis of organ rejection. Other mTOR inhibitors thatare currently marketed or under development include temsirolimus(Torisel™; Wyeth), RAD001 (everolimus; Novartis), MK-8669 (deforolimus;Merck & Ariad pharma), FK506 (tacrolimus; Astellas), TOP216 (ToptargetNS), OSI-027 (OSI Pharma), TAFA93 (Isotechnika), and nab-rapamycin (APPPharma). Many of these mTOR inhibitors are rapamycin or rapamycinderivatives.

In addition to their development as organ transplant rejectionprophylaxis and anti-cancer agents, mTOR inhibitors are also beingdeveloped for the treatment of rheumatoid arthritis, autoimmunedisorders, psoriasis, multiple sclerosis, Parkinson's disease, strokeand peripheral neuropathies. For example, the mTOR inhibitor rapamycinhas been shown to have efficacy in several animal models of autoimmunitysuch as experimental allergic encephalomyelitis, insulin-dependentdiabetes mellitus, murine lupus and adjuvant arthritis, and has beenproposed as a potential therapy in rheumatoid diseases. As to thelatter, antibodies from individuals with rheumatoid or Graves' diseaseactivate fibroblasts through the mTOR pathway, which can be inhibitedwith rapamycin, suggesting that an mTOR blockade would be of value inlimiting inflammation in these diseases. Rapamycin was also found toreduce collagen mRNA levels in human fibroblasts, suggesting that mTORpositively regulates collagen type I synthesis. Thus, mTOR blockade mayalso be of benefit in fibrotic diseases such as scleroderma, wherefibrotic lesions disrupt normal tissue architecture and contribute toorgan failure. Protein expression of the CCR5 chemokine receptorutilized by HIV-1 to enter CD4 T cells and macrophages is also inhibitedby rapamycin, suggesting a clinical utility for blockade ofCCR5-mediated viral entry in immune cells. Recently, rapamycin treatmentwas shown to significantly reduce clinical disease in a patient withdermatomyositis, an autoimmune condition that affects muscle and skin.mTOR inhibitors also have potent neuroprotective and neuroregenerativeproperties in culture and animal models.

However, several adverse reactions have been observed with mTORinhibitors in clinical studies. These include rash, asthenia, mucositis,anorexia, peripheral edema, hypertriglyceridemia, hypertension,hypercholesterolemia, hypercreatinemia, constipation, abdominal pain,diarrhea, headache, fever, urinary tract infection, anemia, nausea,arthralgia, pain, and thrombocytopenia. At least a subset of theseadverse reactions appear to be dose dependent.

BRIEF DESCRIPTION

Methods and pharmaceutical combinations embodying the principles ofembodiments of the invention include co-administering mTOR inhibitorswith a class of compounds generally described as μ-opioid receptorantagonists. The inventors have found that a μ-opioid receptorantagonist, such as methylnaltrexone, can be co-administered with mTORinhibitors in a synergistic manner that permits reducing thetherapeutically effective dosing of the mTOR inhibitors. As noted above,at least a subset of the adverse reactions that are associated with mTORinhibitors appear to be dose-dependent, and thus, the co-administrationof a μ-opioid receptor antagonist allows for the use of decreased dosesof these mTOR inhibitors and the concommitant reduction in the incidenceand/or severity of adverse reactions. The inventors have demonstratedthat methylnaltrexone inhibits the activation of the kinase Akt, whichis an upstream event in the activation of mTOR. Akt and mTOR areinvolved in a complex regulatory feedback loop, and simultaneoustargeting of both Akt and mTOR in accordance with embodiments of theinvention has a synergistic effect.

The use of μ-opioid receptor antagonists in combination with mTORinhibitors may greatly increase the anticancer and immunosuppressiveefficacy of these inhibitors and allow for their use at lower doses,thus lessening the occurrence/severity of adverse effects. Moreover, useof μ-opioid receptor antagonists, such as methylnaltrexone, to increasethe efficacy of these mTOR inhibitors with a resultant decrease indosing required for a therapeutic effect would also greatly decrease thehigh cost of treatment associated with mTOR inhibitors. A method ofimproving the therapeutic index or utility of an mTOR inhibitor byco-administering a synergistically effective amount of an mTOR inhibitorand a μ-opioid receptor antagonist is further contemplated.

Methods embodying the principles of embodiments of the invention includeattenuating, e.g., inhibiting or reducing, cell proliferation andmigration, particularly endothelial cell proliferation and migration,using a combination of mTOR inhibitors and μ-opioid receptorantagonists, including, but not limited to, those that areperipherally-restricted antagonists. According to an aspect of theinvention, a method of treatment is provided that includes administeringto a subject with a disorder characterized by unwanted migration and/orproliferation of cells, a synergistically effective amount of an mTORinhibitor and a μ-opioid receptor antagonist. The treatment may inhibitone or both of migration and proliferation, and the cells may suitablybe endothelial cells, and of particular interest, vascular endothelialcells. Thus, in another embodiment, this disorder characterized byunwanted migration or proliferation of vascular endothelial cells isunwanted angiogenesis. In other words, a method of treating unwantedangiogenesis is contemplated.

According to another aspect, methods of attenuating migration and/orproliferation of cells of a tumor or cancer are provided, includingcontacting the cells with an antimigratory or antiproliferative amountof an mTOR inhibitor and a μ-opioid receptor antagonist. In attenuatingabnormal cell proliferation, activation of the mTOR/Akt signalingpathway is inhibited in a mammal by administering to the mammal asynergistic therapeutically effective amount of an mTOR inhibitor and aμ-opioid receptor antagonist. Thus, in accordance with an embodiment ofthe invention, a method of inhibiting mTOR/Akt pathway signaling incells, e.g., endothelial cells, is also provided, which method includescontacting the cells with a synergistically effective amount of an mTORinhibitor and a μ-opioid receptor antagonist. Another embodiment of theinvention further includes a method of treating cancerous tissue in asubject including, administering to the subject an amount of an mTORinhibitor and a μ-opioid receptor antagonist sufficient to inhibitmTOR/Akt pathway-mediated effects in the cancerous tissue, as well as amethod including contacting a tissue or a population of cells with acomposition or combination including an amount of at least one of anmTOR inhibitor and at least one of a μ-opioid receptor antagonist underconditions effective to synergistically inhibit mTOR/Akt pathway-inducedcell proliferation and migration.

In a further aspect, a method of treating a disorder or diseasecharacterized by hyperproliferation of cells is provided, which methodincludes co-administering to a subject suffering thereof, asynergistically effective amount of an mTOR inhibitor and a μ-opioidreceptor antagonist. Yet another embodiment of the invention includes amethod of treating cancer, e.g., a method of inhibiting growth of atumor in a subject in need thereof, which method includesco-administering a synergistically effective amount of an mTOR inhibitorand a μ-opioid receptor antagonist.

A further embodiment of the invention provides a method of treatingabnormal proliferation of cells expressing a growth factor receptor in amammal by administering to the mammal a synergistic therapeuticallyeffective amount of an mTOR inhibitor and a μ-opioid receptorantagonist. In particular embodiments, the growth factor receptor isvascular endothelial growth factor receptor (VEGF-R), epidermal growthfactor receptor (EGF-R) or insulin-like growth factor receptor (IGF-R).Thus, in accordance with other embodiments of the invention, a method ofinhibiting growth factor signaling in endothelial cells is alsoprovided. In specific embodiments, the growth factor is VEGF, EGF orIGF.

By administering the synergistic combination of mTOR inhibitors andμ-opioid antagonists, in accordance with embodiments of the invention,methods of treatment of autoimmune diseases, psoriasis,neurodegenerative diseases, CCRS-mediated viral entry into immune cells,and nausea and emesis are also contemplated.

Pharmaceutical combinations and packages of mTOR inhibitors and μ-opioidreceptor antagonists as well as accompanying instructions forco-administration are also provided in accordance with embodiments ofthe invention.

mTOR inhibitors for use in the methods in accordance with embodiments ofthe invention generally include compounds that inhibit cell replicationby blocking progression of the cell cycle from G1 to S by inhibitingphosphorylation of serine 389 of p70S6 kinase by mTOR. Known mTORinhibitors include rapamycin and rapamycin derivatives. Several othersmall molecule inhibitors of mTOR activity have also been identified.Use of more than one mTOR inhibitor in the combination therapy inaccordance with embodiments of the inventions is also contemplated.

In some embodiments, the μ-opioid receptor antagonist may be aperipheral μ-opioid receptor antagonist. Peripherally-restrictedμ-opioid receptor antagonists are generally heterocyclic amine compoundsthat also belong to several different classes of compounds. For example,one class is quaternary derivatives of morphinan, and in particular,quaternary derivatives of noroxymorphone. In one embodiment, thequaternary derivative of noroxymorphone is suitably, e.g.,N-methylnaltrexone (or simply methylnaltrexone), N-methylnaloxone,N-methylnalorphine, N-diallylnormorphine, N-allyllevallorphan, orN-methylnalmefene. Another class of peripherally-restricted antagonistsis N-substituted piperidines. In one embodiment, the N-piperidine is apiperidine-N-alkylcarbonylate, such as alvimopan. Other classes ofcompounds that may be of value in embodiments of the invention arequaternary derivatives of benzomorphans, quaternary derivatives ofnormorphanin and polymer conjugates of tertiary derivatives ofmorphanin, benzomorphan and normorphanin.

Embodiments of the invention also encompass administration of more thanone μ-opioid receptor antagonist in therapeutic combinations. Antagonistcombinations may include combinations of μ-antagonists and combinationsof μ- and κ-antagonists, for example, a combination of methylnaltrexoneand alvimopan, or a combination of naloxone and methylnaltrexone.

Other μ-opioid receptor antagonists that may be of use in the methods inaccordance with embodiments of the invention may include tertiaryderivatives of morphinan, and in particular, tertiary derivatives ofnoroxymorphone which include, e.g., naloxone.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention may be better understood and appreciated byreference to the detailed description presented herein in conjunctionwith the accompanying drawings of which:

FIG. 1 depicts the chemical structures of naltrexone andmethylnaltrexone, and the conversion reaction of naltrexone tomethylnaltrexone;

FIG. 2 is an immunoblot demonstrating VEGF-induced phosphorylation(activation) of Akt at serine⁴⁷³ and threonine³⁰⁸ usinganti-phospho-serine⁴⁷³-Akt, anti-phospho-threonine³⁰⁸-Akt and anti-Aktspecific antibodies in human endothelial cells in the presence ofbevacizumab, 5-FU, methylnaltrexone, a combination of bevacizumab andmethylnaltrexone and a combination of 5-FU and methylnaltrexone;

FIG. 3A and FIG. 3B is a graph depicting dose-related effects ofinhibition of VEGF-induced phosphorylation of Akt at serine⁴⁷³ FIG. 3Aand threonine³⁰⁸ FIG. 3B by methylnaltrexone;

FIG. 4 is a graph depicting dose-related effects on inhibition ofVEGF-induced endothelial cell proliferation by rapamycin;

FIG. 5 is a graph depicting dose-related effects on inhibition ofVEGF-induced endothelial cell migration by rapamycin;

FIG. 6 is a graph depicting synergistic inhibition of VEGF-inducedendothelial cell proliferation with a combination of methylnaltrexoneand rapamycin;

FIG. 7 is a graph depicting synergistic inhibition of VEGF-inducedendothelial cell migration with a combination of methylnaltrexone andrapamycin.

FIG. 8 is an immunoblot demonstrating the effect of methylnaltrexone andtemsirolimus on VEGF-induced phosphorylation (activation) of Akt atserine⁴⁷³ and threonine³⁰⁸ using anti-phospho-serine⁴⁷³-Akt,anti-phospho-threonine³⁰⁸-Akt and anti-Akt specific antibodies in humanendothelial cells.

FIG. 9A and FIG. 9B shows immunoblots demonstrating FIG. 9A the effectof methylnaltrexone and temsirolimus on the VEGF-induced formation ofmTOR Complex 1 and mTOR Complex 2 and FIG. 9B the effect of PI3 kinaseinhibition, Src depletion and Rictor depletion on VEGF-inducedphosphorylation (activation) of Akt at serine⁴⁷³ and threonine³⁰⁸ usinganti-phospho-serine⁴⁷³-Akt, anti-phospho-threonine³⁰⁸-Akt and anti-Aktspecific antibodies in human endothelial cells.

FIG. 10A and FIG. 10B provides graphs depicting FIG. 10A synergisticinhibition of VEGF-induced endothelial cell proliferation with acombination of methylnaltrexone and temsirolimus and FIG. 10Bsynergistic inhibition of VEGF-induced endothelial cell migration with acombination of methylnaltrexone and temsirolimus.

FIG. 11A and FIG. 11B provides graphs demonstrating that the synergisticinhibition of endothelial cell FIG. 11A proliferation and FIG. 11Bmigration with a combination of methylnaltrexone and temsirolimus isregulated by tyrosine phosphatase activity.

FIG. 12 is a schematic representation of a molecular basis of thesynergistic activity of methylnaltrexone and mTOR inhibitors.

DETAILED DESCRIPTION

According to the principles manifest in embodiments of the invention,pharmaceutical combinations and methods are provided utilizing acombination of mTOR inhibitors and μ-opioid receptor antagonists.Methods in accordance with embodiments of the invention include treatinga disorder characterized by unwanted and undesired cell proliferationand migration, particularly unwanted and undesired endothelial cellproliferation and/or migration, by co-administering an mTOR inhibitorand a μ-opioid receptor antagonist. As explained in the Examples below,combinations of an mTOR inhibitor and a μ-opioid receptor antagonist,such as methylnaltrexone (MNTX), provide an unexpected synergy inreducing unwanted cell proliferation and migration, e.g., VEGF-inducedproliferation and migration of endothelial cells.

Before explaining at least one embodiment of the invention, it is to beunderstood that the invention is not limited in its application to thedetails set forth in the following description and as exemplified by theExamples. Such description and Examples are not intended to limit thescope of the invention as set forth in the appended claims. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. While the following detailed descriptionand Examples describe the invention through reference to embodimentsutilizing rapamycin, temsirolimus and methylnaltrexone as suitabledrugs, it should be understood that other mTOR inhibitors and μ-opioidreceptor antagonists may also be suitable for use in accordance with theprinciples of the invention.

Further, no admission is made that any reference, including any patentor patent document, cited in this specification constitutes prior art.In particular, it will be understood that, unless otherwise stated,reference to any document herein does not constitute an admission thatany of these documents form part of the common general knowledge in theart in the United States or in any other country. Any discussion of thereferences states what their authors assert, and the applicant reservesthe right to challenge the accuracy and pertinency of any of thedocuments cited herein.

Throughout this disclosure, various aspects of this invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity, andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, as will be understood by one skilled in the art,for any and all purposes, particularly in terms of providing a writtendescription, all ranges disclosed herein also encompass any and allpossible subranges and combinations of subranges thereof, as well as allintegral and fractional numerical values within that range. As anexample, a range of 20% to 40% can be broken down into ranges of 20% to32.5% and 32.5% to 40%, 20% to 27.5% and 27.5% to 40%, etc., all ofwhich are understood to be expressly enumerated in this specification.Any listed range can be easily recognized as sufficiently describing andenabling the same range being broken down into at least equal halves,thirds, quarters, fifths, tenths, etc. As a non-limiting example, eachrange discussed herein can be readily broken down into a lower third,middle third, and upper third, etc.

As will also be understood by one skilled in the art, all language suchas “up to,” “at least,” “greater than,” “less than,” “more than” and thelike include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio. These are only examples of what isspecifically intended. Further, the phrases “ranging/ranges between” afirst indicate number and a second indicate number and “ranging/rangesfrom” a first indicate number “to” a second indicate number are usedherein interchangeably.

Further, the use of “comprising,” “including,” “having,” and variationsthereof herein is meant to encompass the items listed thereafter andequivalents thereof as well as additional items, e.g., other stepsand/or ingredients. These terms encompass the terms “consisting of” and“consisting essentially of.” The use of “consisting essentially of”means that the composition or method may include additional ingredientsand/or steps, but only if the additional ingredients and/or steps do notmaterially alter the basic and novel characteristics of the claimedcomposition or method.

Unless otherwise defined, all scientific and technical terms are usedherein according to conventional usage and have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention belongs. However, as used herein, the following definitionsmay be useful in aiding the skilled practitioner in understanding theinvention:

“Subject” refers to mammals, e.g., humans, mice, dogs, cats, rats.

“Alkyl” refers to a univalent aliphatic hydrocarbon group which issaturated and which may be straight, branched, or cyclic having from 1to about 10 carbon atoms in the chain, and all combinations andsubcombinations of chains therein. Exemplary alkyl groups include, butare not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

“Lower alkyl” refers to an alkyl group having 1 to about 6 carbon atoms.

“Alkenyl” refers to a univalent aliphatic hydrocarbon group containingat least one carbon-carbon double bond and having from 2 to about 10carbon atoms in the chain, and all combinations and subcombinations ofchains therein. Exemplary alkenyl groups include, but are not limitedto, vinyl, propenyl, butynyl, pentenyl, hexenyl, and heptnyl.

“Alkynyl” refers to a univalent aliphatic hydrocarbon group containingat least one carbon-carbon triple bond and having from 2 to about 10carbon atoms in the chain, and combinations and subcombinations ofchains therein. Exemplary alkynyl groups include, but are not limitedto, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

“Alkylene” refers to a divalent aliphatic hydrocarbon group having from1 to about 6 carbon atoms, and all combinations and subcombinations ofchains therein. The alkylene group may be straight, branched, or cyclic.There may be optionally inserted along the alkylene group one or moreoxygen, sulfur, or optionally substituted nitrogen atoms, wherein thenitrogen substituent is an alkyl group as described previously.

“Alkenylene” refers to a divalent alkylene group containing at least onecarbon-carbon double bond, which may be straight, branched, or cyclic.Exemplary alkenylene groups include, but are not limited to, ethenylene(—CH═CH—) and propenylene (—CH═CHCH₂—).

“Cycloalkyl” refers to a saturated monocyclic or bicyclic hydrocarbonring having from about 3 to about 10 carbons, and all combinations andsubcombinations of rings therein. The cycloalkyl group may be optionallysubstituted with one or more cycloalkyl-group substituents. Exemplarycycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

“Acyl” means an alkyl-CO group wherein alkyl is as previously described.Exemplary acyl groups include, but are not limited to, acetyl,propanoyl, 2-methylpropanoyl, butanoyl, and palmitoyl.

“Aryl” refers to an aromatic carbocyclic radical containing from about 6to about 10 carbons, and all combinations and subcombinations of ringstherein. The aryl group may be optionally substituted with one or two ormore aryl group substituents. Exemplary aryl groups include, but are notlimited to, phenyl and naphthyl.

“Aryl-substituted alkyl” refers to a linear alkyl group, preferably alower alkyl group, substituted at a terminal carbon with an optionallysubstituted aryl group, preferably an optionally substituted phenylring. Exemplary aryl-substituted alkyl groups include, for example,phenylmethyl, phenylethyl, and 3(4-methylphenyl)propyl.

“Heterocyclic” refers to a monocyclic or multicyclic ring systemcarbocyclic radical containing from about 4 to about 10 members, and allcombinations and subcombinations of rings therein, wherein one or moreof the members of the ring is an element other than carbon, for example,nitrogen, oxygen, or sulfur. The heterocyclic group may be aromatic ornonaromatic. Exemplary heterocyclic groups include, for example, pyrroleand piperidine groups.

“Halo” refers to fluoro, chloro, bromo, or iodo.

“Co-administration” or “co-administering” is meant to refer to acombination therapy in which two or more agents are administered to apatient or subject by any administration route. Co-administration ofagents may also be referred to as combination therapy or combinationtreatment. The agents may be in the same dosage formulation or separateformulations. For combination treatment with more than one active agent,where the active agents are in separate dosage formulations, the activeagents can be administered concurrently, or they each can beadministered at separately staggered times. The agents of a combinationtreatment may be administered simultaneously or sequentially (e.g., oneagent may directly follow administration of the other or the agents maybe given episodically, e.g., one can be given at one time followed bythe other at a later time, e.g., within a week), as long as they aregiven in a manner sufficient to allow both agents to achieve effectiveconcentrations in the body. The agents may also be administered bydifferent routes, e.g., one agent may be administered intravenouslywhile a second agent is administered intramuscularly, intravenously, ororally.

The terms “peripheral,” or “peripherally-restricted” or“peripherally-acting” in reference to μ-opioid receptor antagonists,designate μ-opioid receptor antagonists that act primarily onphysiological systems and components external to the central nervoussystem. In other words, they exhibit reduced or substantially no centralnervous system (CNS) activity. For example, they do not readily crossthe blood-brain barrier in an amount effective to inhibit the centraleffects of opioids, i.e., they do not effectively inhibit the analgesiceffects of opioids when administered peripherally, that is, they do notreduce the analgesic effect of the opioids. The peripheral μ-opioidreceptor antagonist compounds employed in the embodiments of theinvention suitably exhibit less than about 5-15% of theirpharmacological activity in the CNS, with about 0% (i.e., no) CNSactivity, being most suitable. The non-centrally acting characteristicof a peripheral μ-opioid receptor antagonist is often related to charge,polarity, and/or size of the molecule or species. For example,peripherally-acting quaternary amine μ-opioid receptor antagonists asdescribed herein are positively charged while the central-actingtertiary amine μ-opioid receptor antagonists are neutral molecules.

As used herein, the term “mTOR inhibitor” means a compound or ligand, ora pharmaceutically acceptable salt thereof, which inhibits cellreplication by blocking the progression of the cell cycle from G1 to Sby modulating mTOR activity or expression. The term includes the neutraltricyclic compound rapamycin (sirolimus) and other rapamycin compounds,including, e.g., rapamycin derivatives, rapamycin analogues, and othermacrolide compounds as well as other structurally distinct smallmolecules, e.g. fused bicyclic compounds, that inhibit mTOR activity.These include compounds with a structural similarity to rapamycin, e.g.,compounds with a similar macrocyclic structure that have been modifiedto enhance therapeutic benefit. mTOR inhibitors, including rapamycinderivatives, are described hereinafter.

The terms “treating” or “treatment” used herein include any means ofcontrol of a medical or pathological condition such as prevention, care,relief of the condition, attenuation, alleviation, a reduction of thecondition or symptoms of the condition, and inhibition or arrest ofprogression of the condition.

As used herein, the term “side effect” is meant to refer to an effectother than the purpose or desired effect of a drug. Side effects may bebeneficial or undesirable, i.e., adverse. In the instant case,undesirable effects often occur after the administration of an mTORinhibitor. Such side effects include rash, asthenia, mucositis,anorexia, peripheral edema, hypertriglyceridemia, hypertension,hypercholesterolemia, increased creatinine, constipation, abdominalpain, diarrhea, headache, fever, urinary tract infection, anemia,nausea, arthralgia, pain, and thrombocytopenia.

The term “solvate” refers to a compound provided herein or a saltthereof, which further includes a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Where thesolvent is water, the solvate is a hydrate.

As used herein, the term “potency” refers to the ability or capacity ofan anticancer agent to treat cancer in a subject suffering from cancer.Potency may also be expressed as the dose of a drug required to producea specific effect of a given intensity.

In certain embodiments, the term “unwanted” in connection with cellproliferation and migration, e.g., “unwanted proliferation” or “unwantedmigration,” is meant to refer to “abnormal or pathological ordysregulated or undesirable or inappropriate” proliferation, division,growth or migration of cells that is not part of normal cell turnover,metabolism, growth or propagation, and generally is occurring morerapidly or to a significantly greater extent than typically occurs in anormally functioning cell of the same type and does not serve normalfunction. Unwanted proliferation and unwanted migration is manifest indisorders that are hyperproliferative in nature and include, but are notlimited to, cancers, such as melanoma, lung cancer, breast cancer,pancreatic cancer, prostate cancer, colon cancer or ovarian cancer,psoriasis, rheumatoid arthritis, epidermolytic by perkeratosis,restratosis, restenosis, endometriosis and abnormal wound healing.

In describing an optically active compound, the prefixes R and S areused to denote the absolute configuration of the molecule about itschiral center(s). The symbols (+) and (−) are used to denote the opticalrotation of the compound, i.e., the direction in which a plane ofpolarized light is rotated by the optically active compound. The (−)prefix indicates that the compound is levorotatory, that is, thecompound rotates the plane of polarized light to the left orcounterclockwise. The (+) prefix indicates that the compound isdextrorotatory, that is, the compound rotates the plane of polarizedlight to the right or clockwise. However, the sign of optical rotation,(+) and (−), is not related to the absolute configuration of themolecule, R and S.

In the following description of the methods of the invention, processsteps are carried out at room temperature and atmospheric pressureunless otherwise specified.

In one aspect, an embodiment of the invention relates to methods ofattenuating abnormal or undesirable cellular processes, for example,unwanted cell migration and/or proliferation, particularly unwantedendothelial cell migration and/or proliferation. Methods includeadministering one or more mTOR inhibitors and one or more μ-opioidreceptor antagonists in a synergistically effective amount to cells of,e.g., a tissue or an organ of a patient or subject, particularlyendothelial cells of a tissue or organ of a patient, to inhibit cellmigration and/or proliferation, e.g., endothelial cell migration and/orproliferation.

μ-opioid receptor antagonists have been shown to inhibit unwantedproliferation and migration induced by opioids, endogenous or exogenous,and growth factors, such as VEGF, PDGF, S1P etc. Peripheral μ-opioidreceptor antagonists, in particular, have shown a substantial efficacyin inhibiting opioid and growth factor-induced proliferation andmigration of endothelial cells as disclosed in co-pending U.S. patentapplication Ser. No. 11/908,058. The peripheral μ-opioid receptorantagonist methylnaltrexone (MNTX) inhibits both opioid and growthfactor-induced proliferation and migration in a concentration dependentmanner. Furthermore, it has now been discovered that μ-opioid receptorantagonists, and the peripheral μ-opioid receptor antagonist MNTX inparticular, inhibit agonist-induced endothelial cell (EC) proliferationand migration via inhibition of Akt. The agonists can be opioids,exogenous and/or endogenous, angiogenic factors (e.g., VEGF), and otherproliferation and/or migration stimulating factors (e.g., PDGF, S1P,S1P₃ receptor, RhoA, etc).

μ-opioid receptor antagonists have also been shown to inhibit theability of viruses to infect target cells. Peripheral μ-opioid receptorantagonists, in particular, have shown a substantial efficacy ininhibiting the viral entry of HIV and the expression of CCR5, the cellsurface receptor of this virus, as disclosed in the co-pending U.S.patent application Ser. No. 10/163,482 incorporated herein by reference.

Embodiments of the invention demonstrate that μ-opioid receptorantagonists co-administered with mTOR inhibitors give rise to asignificantly enhanced antiproliferative effect on cancerous cells, andthus provide an increased therapeutic effect, e.g., administeringperipheral μ-opioid receptor antagonists to certain tumors canpotentiate tumor response to mTOR inhibitors. As illustrated in theExamples below, a significantly increased antiproliferative andantimigratory effect is obtained with the above disclosedco-administered combinations utilizing lower concentrations of the mTORinhibitor and the μ-opioid receptor antagonist compared to the treatmentregimes in which the drugs are used alone. For example,co-administration of a μ-opioid receptor antagonist with an mTORinhibitor in accordance with embodiments of the invention may reduce thedose of the mTOR inhibitor or increase potency or efficacy or both.Further, the co-administration of a μ-opioid receptor antagonist and anmTOR inhibitor in accordance with embodiments of the invention may alsohave prophylactic value.

There is also the potential to provide therapy wherein adverse sideeffects associated with mTOR inhibitors are considerably reducedcompared to those normally observed with the mTOR inhibitors used alonein larger doses. These side effects include rash, asthenia, mucositis,anorexia, peripheral edema, hypertriglyceridemia, hypertension,hypercholesterolemia, increased creatinine, constipation, abdominalpain, diarrhea, headache, fever, urinary tract infection, anemia,nausea, arthralgia, pain, and thrombocytopenia. At least a subset ofthese adverse reactions are dose dependent. The potential for lowerdosing is achieved utilizing embodiments in accordance with theinvention.

There is the further potential to provide therapy wherein the high costsassociated with mTOR inhibitor therapy are considerably reduced comparedto those normally observed with the mTOR inhibitors used alone in largerdoses. In addition, the co-administration of a μ-opioid receptorantagonist with an mTOR inhibitor can considerably increase the efficacyand potency of the mTOR inhibitor compared to that normally observedwith the mTOR alone.

Methods embodying the principles manifest in embodiments of theinvention include attenuating, e.g., inhibiting or reducing, unwantedcell proliferation and migration, particularly endothelial cellproliferation and migration, using mTOR inhibitors and μ-opioid receptorantagonists, including, but not limited to, those that areperipherally-restricted antagonists. According to one aspect of theinvention, a method of treatment is provided that involves administeringto a subject with a disorder characterized by unwanted migration orproliferation of endothelial cells, a synergistically effective amountof an mTOR inhibitor and a μ-opioid receptor antagonist. The treatmentmay inhibit one or both of migration and proliferation. In a furtherembodiment, the unwanted migration or proliferation is unwantedmigration or proliferation of vascular endothelial cells that is treatedwith a synergistic amount of an mTOR inhibitor and a μ-opioid receptorantagonist. In another embodiment, the disorder characterized byunwanted migration or proliferation of vascular endothelial cells isunwanted angiogenesis. Thus, a method of treating unwanted angiogenesisis contemplated.

According to yet another aspect, methods of attenuating migration and/orproliferation of cells of a tumor or cancer are provided, which methodsinclude contacting the cells with an antimigratory or antiproliferativeamount of mTOR inhibitor and a μ-opioid receptor antagonist. Inattenuating cell proliferation, a method of treating abnormal cellproliferation of cells that exhibit increased activation of the mTOR/Aktsignaling pathway in a mammal is provided, which method includesadministering to the mammal a synergistic therapeutically effectiveamount of an mTOR inhibitor and a μ-opioid receptor antagonist. Thus, amethod of inhibiting mTOR/Akt pathway signaling in endothelial cells isalso provided. Methods involve contacting the cells with asynergistically effective amount of an mTOR inhibitor and a μ-opioidreceptor antagonist. A method of treating cancerous tissue in a subjectis also provided, which method includes administering to the subject anamount of an mTOR inhibitor and a μ-opioid receptor antagonistsufficient to inhibit mTOR/Akt pathway-mediated effects in the canceroustissue, as well as a method including contacting a tissue or apopulation of cells with a composition or combination including anamount of at least one of an mTOR inhibitor and at least one of aμ-opioid receptor antagonist under conditions effective to inhibitmTOR/Akt pathway-induced proliferation and migration.

In yet another embodiment, a method of treating abnormal proliferationof cells that express a growth factor receptor in a mammal is provided,which method includes administering to the mammal a synergistictherapeutically effective amount of an mTOR inhibitor and a μ-opioidreceptor antagonist. In particular embodiments, the growth factorreceptor is vascular endothelial growth factor receptor (VEGF-R),epidermal growth factor receptor (EGF-R) or insulin-like growth factorreceptor (IGF-R). Thus, a method of inhibiting growth factor signalingin endothelial cells is also provided. In specific embodiments, thegrowth factor is VEGF, EGF or IGF. Methods involve contacting the cellswith a synergistically effective amount of an mTOR inhibitor and aμ-opioid receptor antagonist. A method of treating cancerous tissue in asubject is also provided, which method includes administering to thesubject an amount of an mTOR inhibitor and a μ-opioid receptorantagonist sufficient to inhibit growth factor-induced effects in thecancerous tissue, as well as a method including contacting a tissue or apopulation of endothelial cells with a composition or combinationincluding an amount of at least one of an mTOR inhibitor and at leastone of a μ-opioid receptor antagonist under conditions effective toinhibit growth factor-induced proliferation and migration. It isparticularly contemplated that these effects in cancerous tissue areVEGF-, EGF- or IGF-induced.

In yet a further embodiment, a method of treating abnormal cellproliferation of cells that express a hormone receptor in a mammal isprovided, which method includes administering to the mammal asynergistic therapeutically effective amount of an mTOR inhibitor and aμ-opioid receptor antagonist. In particular embodiments, the hormonereceptor is estrogen receptor (ER), progesterone receptor (PR) orandrogen receptor (AR). Thus, a method of inhibiting hormone signalingin endothelial cells is also provided. In specific embodiments, thehormone is estrogen, progesterone or androgen. Methods involvecontacting the cells with a synergistically effective amount of an mTORinhibitor and a μ-opioid receptor antagonist. A method of treatingcancerous tissue in a subject is also provided, which method includesadministering to the subject an amount of an mTOR inhibitor and aμ-opioid receptor antagonist sufficient to inhibit hormone-inducedeffects in the cancerous tissue, as well as a method includingcontacting a tissue or a population of endothelial cells with acomposition or combination including an amount of at least one of anmTOR inhibitor and at least one of a μ-opioid receptor antagonist underconditions effective to inhibit hormone-induced proliferation andmigration. It is particularly contemplated that these effects incancerous tissue are estrogen-, progesterone- or androgen-induced.

In yet a further aspect, a method of treating a disorder or diseasecharacterized by hyperproliferation of cells is provided, which methodincludes co-administering to a subject suffering thereof asynergistically effective amount of an mTOR inhibitor and a μ-opioidreceptor antagonist. Yet another embodiment of the invention is a methodof treating cancer in a subject in need thereof, comprisingco-administering a synergistically effective amount of an mTOR inhibitorand a μ-opioid receptor antagonist. A further embodiment of theinvention is a method of inhibiting growth of a tumor in a subject inneed thereof, comprising co-administering a synergistically effectiveamount of an mTOR inhibitor and a μ-opioid receptor antagonist.

In a further aspect, a method of treating an autoimmune disease in apatient is provided, which method includes co-administering asynergistically effective amount of an mTOR inhibitor and a μ-opioidreceptor antagonist. Autoimmune diseases of the invention include, butare not limited to, allergic encephalomyelitis, insulin-dependentdiabetes mellitus, lupus, rheumatoid arthritis, multiple sclerosis,dermatomyositis, Grave's disease and adjuvant arthritis.

Yet another aspect of the invention provides a method of treatingpsoriasis in a patient, including co-administering a synergisticallyeffective amount of an mTOR inhibitor and a μ-opioid receptorantagonist. In a particular embodiment, the synergistically effectiveamount of an mTOR inhibitor and a μ-opioid receptor antagonist isapplied topically.

A further embodiment of the invention is a method of treating aneurodegenerative disease, which method includes the co-administrationof a synergistically effective amount of an mTOR inhibitor and aμ-opioid receptor antagonist. In particular embodiments, theneurodegenerative disease is Parkinson's disease or multiple sclerosis.

Yet a further embodiment of the invention is a method of inhibitingCCR5-mediated viral entry into immune cells which includes theco-administration of a synergistically effective amount of an mTORinhibitor and a μ-opioid receptor antagonist. The inhibition ofCCR5-mediated HIV entry into immune cells is particularly contemplated.Further, a method of treating HIV/AIDS which includes co-administeringto a subject suffering thereof a synergistically effective amount of anmTOR inhibitor and a μ-opioid receptor antagonist is provided.

In another aspect, nausea and emesis, induced by treatment of cancerwith mTOR inhibitor, may be alleviated by co-administering a μ-opioidreceptor antagonist, such as the peripheral μ-opioid receptor antagonistmethylnaltrexone.

As noted above, mTOR inhibitors include compounds or ligands, orpharmaceutically acceptable salts thereof, which inhibit cellreplication by blocking the progression of the cell cycle from G1 to Sthrough the modulation of mTOR activity or expression. mTOR inhibitorsthat are currently available or under development, include temsirolimus(Torisel™; Wyeth), RAD001 (everolimus; Novartis), MK-8669 (deforolimus;Merck & Ariad pharma), TOP216 (Toptarget NS), OSI-027 (OSI Pharma),TAFA93 (Isotechnika), nab-rapamycin (APP Phama) and tacrolimus (FK506;Astellas).

mTOR inhibitors include rapamycin and related compounds. Rapamycin is amacrolide produced by Streptomyces. Rapamycins are potentimmunosuppressive agents and are used clinically to prevent rejection oftransplanted organs. Rapamycin and related compounds are also currentlyunder development as anti-cancer therapeutic agents. The rapamycinsuseful in embodiments of the invention include compounds that arechemically or biologically modified as derivatives of the rapamycinnucleus, while still retaining immunosuppressive or anti-cancerproperties. Accordingly, rapamycins include rapamycin itself, andesters, ethers, carbamates, oximes, hydrazones, and hydroxylamines ofrapamycin, as well as rapamycins in which functional groups on therapamycin nucleus have been modified, for example through reduction oroxidation.

Specifically, the structure of rapamycin is given as formula (A) shownbelow:

Many of the rapamycin derivatives disclosed above in current use ordevelopment have the basic rapamycin structure with substitutions at theC-40 position. If the substituent at position 40 is designated as R,then the following substitutions and corresponding compounds are:R=—OP(O)(Me)₂, AP23573 (International Patent Publication Nos. WO98/02441 and WO 2001/14387); R=—OC(O)C(CH₃)(CH₂OH), temsirolimus (U.S.Pat. No. 5,362,718); R=—OCH₂CH₂OH, everolimus (U.S. Pat. No. 5,665,772);R=—OCH₂CH₂OEt, biolimus; R=-tetrazole, ABT-578 (International PatentPublication No. WO 99/15530). All patents and applications are herebyincorporated by reference.

Many other rapamycin derivatives include substitutions in the C-40and/or C-16 and/or C-32 positions. Esters and ethers of rapamycin aredescribed in the following patents, which are all hereby incorporated byreference: alkyl esters (U.S. Pat. No. 4,316,885); aminoalkyl esters(U.S. Pat. No. 4,650,803); fluorinated esters (U.S. Pat. No. 5,100,883);amide esters (U.S. Pat. No. 5,118,677); carbamate esters (U.S. Pat. Nos.5,118,678; 5,411,967; 5,434,260; 5,480,988; 5,480,989; 5,489,680); silylesters (U.S. Pat. No. 5,120,842); aminodiesters (U.S. Pat. No.5,162,333); sulfonate and sulfate esters (U.S. Pat. No. 5,177,203);esters (U.S. Pat. No. 5,221,670); alkoxyesters (U.S. Pat. No.5,233,036); O-aryl, -alkyl, -alkenyl, and -alkynyl ethers (U.S. Pat. No.5,258,389); carbonate esters (U.S. Pat. No. 5,260,300); arylcarbonyl andalkoxycarbonyl carbamates (U.S. Pat. No. 5,262,423); carbamates (U.S.Pat. No. 5,302,584); hydroxyesters (U.S. Pat. No. 5,362,718); hinderedesters (U.S. Pat. No. 5,385,908); heterocyclic esters (U.S. Pat. No.5,385,909); gem-disubstituted esters (U.S. Pat. No. 5,385,910); aminoalkanoic esters (U.S. Pat. No. 5,389,639); phosphorylcarbamate esters(U.S. Pat. No. 5,391,730); amidino carbamate esters (U.S. Pat. No.5,463,048); hindered N-oxide esters (U.S. Pat. No. 5,491,231); biotinesters (U.S. Pat. No. 5,504,091); O-alkyl ethers (U.S. Pat. No.5,665,772); and PEG esters of rapamycin (U.S. Pat. No. 5,780,462);32-esters and ethers (U.S. Pat. No. 5,256,790). The preparation of theseesters and ethers is disclosed in the patents listed above. All patentsand applications are hereby incorporated by reference.

Also included are oximes, hydrazones, and hydroxylamines of rapamycin asdisclosed in U.S. Pat. Nos. 5,373,014, 5,378,836, 5,023,264, and5,563,145. The preparation of these oximes, hydrazones, andhydroxylamines is disclosed in the above-listed patents. The preparationof 40-oxorapamycin is disclosed in U.S. Pat. No. 5,023,263. All thesepatents are hereby incorporated by reference.

Other small molecule inhibitors of mTOR include fused bicyclic compounds(International Patent Publication Nos. WO 2007/61737, WO 2007/87395 andWO 2007/64993), heteroaromatic amines (International Patent PublicationNo. WO 2001/19828), pyrrolopyrimidine compounds (International PatentPublication No. WO 2005/47289), diphenyl-dihydro-indol-2-one derivatives(International Patent Publication No. WO 2005/97107), andtrimethy-dodeca-triene derivatives (US Patent Publication No.2007/037887). All of these patents are hereby incorporated by reference.

The μ-opioid receptor antagonists in accordance with embodiments of theinvention may include both centrally and peripherally acting μ-opioidreceptor antagonists. However, it is contemplated that those antagonistsof particular value are suitably the peripherally-restricted μ-opioidreceptor antagonists.

μ-opioid receptor antagonists form a class of compounds that can vary instructure while maintaining their antagonist properties. These compoundsinclude tertiary and quaternary morphinans, in particular noroxymorphonederivatives, N-substituted piperidines, and in particular,piperidine-N-alkylcarboxylates, and tertiary and quaternarybenzomorphans, and tertiary and quaternary normorphinan derivatives, inparticular 6-corboxy-normorphinan derivatives. Tertiary compoundantagonists are fairly lipid soluble and cross the blood-brain barriereasily. Examples of μ-opioid receptor antagonists that cross theblood-brain barrier and are centrally (and peripherally) active include,e.g., naloxone (which is commercially available from BaxterPharmaceutical Products, Inc.), and nalmefene (available, e.g., fromDuPont Pharma). Peripherally-restricted antagonists, on the other hand,are typically charged, polar, and/or of high molecular weight, each ofwhich impedes their crossing the blood-brain barrier. Methylnaltrexoneis a quaternary derivative of the tertiary μ-opioid receptor antagonist,naltrexone. Addition of the methyl group to naltrexone forms a compoundwith greater polarity and lower lipid solubility. Thus, methylnaltrexonedoes not cross the blood-brain barrier and has the potential forblocking the undesired adverse effects which are typically mediated byperipherally located receptors.

A peripheral μ-opioid receptor antagonist for use in embodiments of theinvention may be a compound which is a quaternary morphinan derivative,and in particular, a quaternary noroxymorphone of formula (I):

a single enantiomer, a mixture of enantiomers, an individualdiastereomer, or a mixture of diastereomers thereof; or apharmaceutically acceptable salt, solvate, or prodrug thereof, wherein Ris alkyl, alkenyl, alkynyl, aryl, cycloalkyl-substituted alkyl, orarylsubstituted alkyl, and X⁻ is an anion, especially a chloride,bromide, iodide, carbonate or methylsulfate anion. The noroxymorphonederivatives of formula (I) can be prepared, for example, according tothe procedure in U.S. Pat. No. 4,176,186, which is incorporated hereinby reference; see also, U.S. Pat. Nos. 4,719,215; 4,861,781; 5,102,887;5,972,954; and 6,274,591; U.S. Patent Application Nos. 2002/0028825 and2003/0022909; and PCT publication Nos. WO 99/22737 and WO 98/25613, allof which are hereby incorporated by reference.

A compound of formula (I) of particular value is N-methylnaltrexone (orsimply methylnaltrexone), wherein R is cyclopropylmethyl as representedin formula (II):

a single enantiomer, a mixture of enantiomers, an individualdiastereomer, or a mixture of diastereomers thereof; or apharmaceutically acceptable salt, solvate, or prodrug thereof, whereinX⁻ is as described above. Methylnaltrexone is a quaternary derivative ofthe μ-opioid receptor antagonist naltrexone. Methylnaltrexone exists asa salt (e.g., N-methylnaltrexone bromide) and the terms“methylnaltrexone” or “MNTX”, as used herein, therefore embrace suchsalts. “Methylnaltrexone” or “MNTX” thus specifically includes, but isnot limited to, bromide salts, chloride salts, iodide salts, carbonatesalts, and methylsulfate salts of methylnaltrexone. Names used for thebromide salt of MNTX in the literature, for example, include:methylnaltrexone bromide; N-methylnaltrexone bromide; naltrexonemethobromide; naltrexone methyl bromide; SC-37359; MRZ-2663-BR; andN-cyclopropylmethylnoroxy-morphine-methobromide. Thus, herein, the term“methylnaltrexone” should be understood to mean any appropriate form ofmethylnaltrexone, e.g., N-methylnaltrexone or any pharmaceuticallyacceptable salt thereof, any prodrug thereof, enantiomers thereof,epimers thereof (the latter of which are described hereinafter).

The compounds of formulas (I) and (II), e.g., methylnaltrexone, may havechiral centers and can, therefore, occur as stereochemical isomers byvirtue of the substituent placement on those chiral centers. Suchstereochemical isomers, e.g., enantiomers, diastereomers, are within thescope of the compounds contemplated for use in embodiments of theinvention. In the compositions and methods of embodiments of theinvention, compounds employed may be individual stereoisomers, as wellas mixtures of stereoisomers, e.g., mixtures of enantiomers, mixtures ofdiastereomers. In certain aspects, methods are provided that utilizecompounds which are substantially pure stereoisomers. All tautomers arealso intended to be encompassed within the compositions of theinvention.

For example, the R and S configurations of methynaltrexone are known,and in some embodiments, isolated R—N isomers of methylnaltrexone may beutilized in formulations and methods. As used herein, the designation of“R—N-isomer” of methylnaltrexone refers to such compounds in the (R)configuration with respect to the nitrogen. Isolated isomer compoundsinclude, but are not limited to, R—N isomer methylnaltrexone compoundsdescribed in U.S. patent application Ser. No. 11/441,395, and PatentCooperation Treaty published application WO2006/127899, incorporatedherein by reference. In some embodiments, the active compound is an R—Nisomer methylnaltrexone, or a salt thereof. The R—N isomer ofmethylnaltrexone, described in U.S. Ser. No. 11/441,395, is an opioidantagonist.

In some embodiments, isolated S—N isomers of methylnaltrexone may beutilized in formulations and methods. As used herein, the designation of“S—N-isomer” of methylnaltrexone refers to such compounds in the (S)configuration with respect to the nitrogen. Isolated isomer compoundsinclude, but are not limited to, S—N isomer of methylnaltrexonecompounds described in U.S. patent application Ser. No. 11/441,452, andPatent Cooperation Treaty published application WO2006/127898,incorporated herein by reference. In some embodiments, the activecompound is an S—N isomer methylnaltrexone, or a salt thereof. The S—Nisomer of methylnaltrexone, described in U.S. Ser. No. 11/441,452, is anopioid agonist.

In certain embodiments, the methylnaltrexone utilized in formulations ordosage preparations described herein is a mixture of stereoisomerscharacterized in that it has an overall opioid antagonistic effect. Forexample, the methylnaltrexone may be a mixture of R—N and S—Nmethylnaltrexone such that a mixture itself acts as an antagonist andwould be useful for methods of use described herein for opioidantagonists. In certain embodiments, R—N methylnaltrexone is used whichis substantially free of S—N methylnaltrexone.

In certain embodiments of the invention, at least about 99.6%, 99.7%,99.8%, 99.85%, 99.9%, or 99.95% of methylnaltrexone is in the (R)configuration with respect to nitrogen. Methods for determining theamount of (R)—N-isomer, present in a sample as compared to the amount of(S)—N-isomer present in that same sample, are described in detail inWO2006/127899, the entirety of which is hereby incorporated herein byreference. In other embodiments, methylnaltrexone contains 0.15%, 0.10%,or less (S)—N-isomer.

It will be understood by those skilled in the art that, where referenceis made herein to amounts of methylnaltrexone utilized in formulations,dosage preparations, or methods, those amounts may refer to total amountof methylnaltrexone (or salt thereof), or to amount of relevant activeform of methylnaltrexone for a particular purpose (e.g., opioidantagonism), whether or not other forms of methylnaltrexone are alsopresent. Furthermore, as indicated herein, dosages or amounts aresometimes defined with reference to a particular form ofmethylnaltrexone (e.g., N-methylnaltrexone bromide). Where a differentform or salt of methylnaltrexone is used, those of ordinary skill in theart will appreciate that such dosages or amounts may be adjusted to adose or amount that provides an equivalent amount of activemethylnaltrexone.

Methylnaltrexone is commercially available from, e.g., MallinckrodtPharmaceuticals, St. Louis, Mo. Methylnaltrexone is provided as a whitecrystalline powder, freely soluble in water, typically as the bromidesalt. The compound as provided is 99.4% pure by reverse phase HPLC, andcontains less than 0.011% unquaternized naltrexone by the same method.Methylnaltrexone can be prepared as a sterile solution at aconcentration of, e.g., about 5 mg/mL.

Other peripheral μ-opioid receptor antagonists may include N-substitutedpiperidines, and in particular, piperidine-N-alkylcarboxylates asrepresented by formula (III):

wherein R¹ is hydrogen or alkyl; R² is hydrogen, alkyl, or alkenyl; R³is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl; R⁴ is hydrogen, alkyl, or alkenyl; A is OR⁵ orNR⁶R⁷; wherein R⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl; R⁶ is hydrogen or alkyl; R⁷ is hydrogen, alkyl,alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,cycloalkenyl-substituted alkyl or aryl-substituted alkyl, oralkylene-substituted B or together with the nitrogen atom to which theyare attached, R⁶ and R⁷ form a heterocyclic ring selected from pyrroleand piperidine; B is

wherein R⁸ is hydrogen or alkyl; R⁹ is hydrogen, alkyl, alkenyl, aryl,cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,cycloalkenyl-substituted alkyl or aryl-substituted alkyl or togetherwith the nitrogen atom to which they are attached, R⁸ and R⁹ form aheterocyclic ring selected from pyrrole and piperidine; W is OR¹⁰,NR¹¹R¹², or OE; wherein R¹⁰ is hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substitutedalkenyl, or aryl-substituted alkyl; R¹¹ is hydrogen or alkyl; R¹² ishydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl,aryl-substituted alkyl, or alkylene-substituted C(═O)Y or, together withthe nitrogen atom to which they are attached, R¹¹ and R¹² form aheterocyclic ring selected from pyrrole and piperidine;

E is

alkylene-substituted (C═O)D, or —R¹³OC(═O)R¹⁴; wherein R¹³ isalkyl-substituted alkylene; R¹⁴ is alkyl; D is OR¹⁵ or NR¹⁶R¹⁷; whereinR¹⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl substituted alkyl, oraryl-substituted alkyl; R¹⁶ is hydrogen, alkyl, alkenyl, aryl,aryl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkyl substitutedalkyl, or cycloalkenyl-substituted alkyl; R¹⁷ is hydrogen or alkyl or,together with the nitrogen atom to which they are attached, R¹⁶ and R¹⁷form a heterocyclic ring selected from the group consisting of pyrroleor piperidine;

Y is OR¹⁸ or NR¹⁹R²⁰; wherein R¹⁸ is hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R¹⁹ ishydrogen or alkyl; R²⁰ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl,cycloalkenyl, cycloalkylsubstituted alkyl, cycloalkenyl-substitutedalkyl, or aryl-substituted alkyl or, together with the nitrogen atom towhich they are attached, R¹⁹ and R²⁰ form a heterocyclic ring selectedfrom pyrrole and piperidine; R²¹ is hydrogen or alkyl; and n is 0 to 4.

Particular piperidine-N-alkylcarbonylates which may be of value areN-alkylamino-3,4,4 substituted piperidines, such as alvimopanrepresented below as formula (IV):

N-substituted piperidines may be prepared as disclosed in U.S. Pat. Nos.5,270,328; 6,451,806; 6,469,030, all of which are hereby incorporated byreference. Alvimopan is available from Adolor Corp., Exton, Pa. Suchcompounds have moderately high molecular weights, a zwitterion form, anda polarity that prevent penetration of the blood-brain barrier.

Still other peripheral μ-opioid receptor antagonist compounds mayinclude quaternary benzomorphan compounds. Quaternary benzomorphancompounds, which may be employed in embodiments of the invention, havethe following formula (V):

wherein R¹ is hydrogen, acyl, or acetoxy; and R² is alkyl or alkenyl; Ris alkyl, alkenyl, or alkynyl and X⁻ is an anion, especially a chloride,bromide, iodide, or methylsulfate anion.

Specific quaternary derivatives of benzomorphan compounds that may beemployed in embodiments of the invention include the following compoundsof formula (V):2′-hydroxy-5,9-dimethyl-2,2-diallyl-6,7-benzomorphanium-bromide;hydroxy-5,9-dimethyl-2-n-propyl-2-allyl-6,7-benzomorphanium-bromide;2′-hydroxy-5,9-dimethyl-2-n-propyl-2-propargyl-6,7-benzomorphanium-bromide;and2′-acetoxy-5,9-dimethyl-2-n-propyl-2-allyl-6,7-benzomorphanium-bromide.

Other quaternary benzomorphan compounds that may be employed inembodiments of the invention are described, for example, in U.S. Pat.No. 3,723,440, the entire disclosure of which is incorporated herein byreference.

Other peripheral opioid antagonists may include 6-carboxy-normorphinanderivatives, particularly N-methyl-C-normorphinan derivatives, asdescribed in U.S. Pat. No. 7,501,434, entitled “6-Carboxy-NormorphinanDerivatives, Synthesis and Uses Thereof,” incorporated in its entiretyherein by reference and including the compound having the followingformula (VI):

Peripheral opioid antagonists may also include polymer conjugates ofopioid antagonists, as described in U.S. patent application Ser. No.11/332,964, hereby incorporated by reference. Specific polymerconjugates include PEGylated naloxone and naltrexone.

Embodiments of the invention also encompass administration of more thanone μ-opioid receptor, including combinations of μ-opioid receptorantagonists and combinations of mu and kappa antagonists, for example, acombination of methylnaltrexone and alvimopan.

The compounds employed in embodiments of the invention may exist inprodrug form. As used herein, “prodrug” is intended to include anycovalently bonded carriers which release the active parent drug orcompounds that are metabolized in vivo to an active drug or othercompounds employed in embodiments of the invention in vivo when suchprodrug is administered to a mammalian subject. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compoundsemployed in some embodiments of the invention may, if desired, bedelivered in prodrug form. Thus, embodiments of the inventioncontemplate methods of delivering prodrugs. Prodrugs of the compoundsemployed in embodiments of the invention may be prepared by modifyingfunctional groups present in the compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, amino, or carboxy group is bonded to any group that,when the prodrug is administered to a mammalian subject, cleaves to forma free hydroxyl, free amino, or carboxylic acid, respectively. Otherexamples include, but are not limited to, acetate, formate, and benzoatederivatives of alcohol and amine functional groups; and alkyl,carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl,iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl,benzyl, and phenethyl esters, and the like.

As noted, the compounds employed in embodiments of the invention may beprepared in a number of ways well known to those skilled in the art. Allpreparations disclosed in embodiments of the invention are contemplatedto be practiced on any scale, including milligram, gram, multigram,kilogram, multikilogram, or commercial pharmaceutical scale.

As noted above for methylnaltrexone, compounds employed in embodimentsof the invention may contain one or more asymmetrically-substitutedcarbon atoms, and may be isolated in optically active or racemic form.Thus, all chiral, diastereomeric, racemic form, epimeric form, and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated. Itis well known in the art how to prepare and isolate such opticallyactive forms. For example, mixtures of stereoisomers may be separated bystandard techniques including, but not limited to, resolution of racemicform, normal, reverse-phase, and chiral chromatography, preferentialsalt formation, recrystallization, and the like, or by chiral synthesiseither from chiral starting materials or by deliberate synthesis oftarget chiral centers.

Methods of embodiments of the invention, generally speaking, may bepracticed using any mode of administration that is medically acceptable,e.g., any mode that produces effective levels of the active compoundswithout causing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, topical (as by powder, ointment,drops, transdermal patch, or iontophoretic device), transdermal,sublingual, intramuscular, infusion, intravenous, pulmonary,intramuscular, intracavity, as an aerosol, aural (e.g., via eardrops),intranasal, inhalation, intraocular, or subcutaneous.

Additionally, the compounds in accordance with embodiments of theinvention may be administered as an enterically coated tablet orcapsule. In some embodiments, the μ-opioid receptor antagonist isadministered by a slow infusion method or by a time-release orcontrolled-release method or as a lyophilized powder.

Further, the compounds in accordance with embodiments of the inventionmay be administered topically. Formulations for topical administrationmay include ointments, lotions, creams, gels, drops, suppositories,sprays, liquids and powders. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be necessaryor desirable.

When administered, the compounds of embodiments of the invention aregiven in pharmaceutically acceptable amounts and in pharmaceuticallyacceptable compositions or preparations. Such preparations may routinelycontain salts, buffering agents, preservatives, and optionally othertherapeutic ingredients.

When used in medicine, pharmaceutically acceptable salts of thecompounds in accordance with embodiments of the invention may be used,but non-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic,tartaric, citric, methanesulfonic, formic, succinic,naphthalene-2-sulfonic, pamoic, 3-hydroxy-2-naphthalenecarboxylic, andbenzene sulfonic.

Buffering agents and preservatives may also be included in preparationsin accordance with embodiments of the invention. Suitable bufferingagents may include, but are not limited to, acetic acid and saltsthereof (1-2% w/v); citric acid and salts thereof (1-3% w/v); boric acidand salts thereof (0.5-2.5% w/v); and phosphoric acid and salts thereof(0.8-2% w/v). Suitable preservatives may include, but are not limitedto, benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v); and thimerosal (0.004-0.02% w/v).

For ease of administration, a pharmaceutical composition in accordancewith embodiments of the invention may also contain one or morepharmaceutically acceptable excipients, such as lubricants, diluents,binders, carriers, and disintegrants. Other auxiliary agents mayinclude, e.g., stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, coloring, flavoring, and/or aromaticactive compounds.

A pharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material, orformulation auxiliary of any type. For example, suitablepharmaceutically acceptable carriers, diluents, solvents, or vehiclesinclude, but are not limited to, water, salt (buffer) solutions,alcohols, gum arabic, mineral and vegetable oils, benzyl alcohols,polyethylene glycols, gelatin, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin,vegetable oils, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxyl methylcellulose, polyvinylpyrrolidone, etc. Proper fluidity may be maintained, for example, by theuse of coating materials such as lecithin or by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. Prevention of the action of microorganisms may be ensuredby the inclusion of various antimicrobial, e.g., antibacterial andantifungal, agents such as paraben, chlorobutanol, phenol, sorbic acidand the like.

If a pharmaceutically acceptable solid carrier is used, the dosage formof the compounds suitable for use in embodiments of the invention may betablets, capsules, powders, suppositories, or lozenges. If a liquidcarrier is used, soft gelatin capsules, transdermal patches, aerosolsprays, topical cream, syrups or liquid suspensions, emulsions, orsolutions may be the dosage form.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably nonaqueous or aqueous solutions, as wellas dispersions, suspensions, emulsions, or implants, includingsuppositories. Ampoules are often convenient unit dosages. Injectabledepot-form may also be suitable and may be made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters), and poly(anhydrides).Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules such as soft gelatincapsules. A syrup, elixir, or the like can be used wherein a sweetenedvehicle is employed.

As noted, other pharmaceutical delivery systems may includetime-release, delayed-release, or sustained-release delivery systems.Such systems can avoid repeated administrations of the compounds of theinvention, increasing convenience to the patient and the physician andmaintaining sustained plasma levels of compounds. Many types ofcontrolled-release delivery systems are available and known to those ofordinary skill in the art.

For example, compounds of embodiments of the invention may be combinedwith pharmaceutically acceptable sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions. Asustained-release matrix, as used herein, is a matrix made of materials,usually polymers, which are degradable by enzymatic or acid-basehydrolysis or by dissolution. Once inserted into the body, the matrix isacted upon by enzymes and body fluids. A sustained-release matrix may bedesirably chosen from biocompatible materials such as liposomes;polymer-based system such as polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(copolymers of lactic acid and glycolic acid), polyanhydrides,poly(ortho)esters, polysaccharides, polyamino acids, hyaluronic acid,collagen, chondroitin sulfate, polynucleotides, polyvinyl propylene,polyvinyl pyrrolidone, and silicone; nonpolymer system such ascarboxylic acids, fatty acids, phospholipids, amino acids, and lipidssuch as sterols; hydrogel release systems; silastic systems;peptide-based systems; implants and the like. Specific examples include,but are not limited to: (a) an erosional system in which thepolysaccharide is contained in a form within a matrix, found in U.S.Pat. Nos. 4,452,775, 4,675,189, and 5,736,152 (herein incorporated byreference in their entireties), and (b) a diffusional system in which anactive component permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974, and 5,407,686 (hereinincorporated by reference in their entireties). In addition, apump-based hard-wired delivery system can be used, some of which areadapted for implantation. Suitable enteric coatings are described in PCTpublication No. WO 98/25613 and U.S. Pat. No. 6,274,591, bothincorporated herein by reference. Sustained- or controlled-releasecompositions may also be formulated as those wherein the active compoundis protected with differentially degradable coatings, such as bymicroencapsulation, multiple coatings, etc.

Respecting methylnaltrexone specifically, aqueous formulations mayinclude a chelating agent, a buffering agent, an anti-oxidant and,optionally, an isotonicity agent, preferably pH adjusted to between 3.0and 3.5. Formulations that are stable to autoclaving and long termstorage are described in U.S. patent application Ser. No. 10/821,811,published as 2004/0266806, entitled “Pharmaceutical Formulation,” thedisclosure of which is incorporated herein by reference. Formulations ofmethylnaltrexone with increased shelf-life are also described inInternational Patent Publication No. WO 2008/19115, entitled“Formulations for Parenteral Delivery of Compounds and Uses Thereof,”hereby incorporated by reference. Lyophilized formulations ofmethylnaltrexone are described in U.S. patent application Ser. No.11/899,724 and formulations comprising particles containingmethylnaltrexone are described in U.S. Pat. No. 6,419,959, which isincorporated herein by reference. Formulations suitable for transdermaldelivery of methylnaltrexone are described in International PatentPublication No. 2007/41544, hereby incorporated by reference.

Compounds in accordance with embodiments of the invention, mTORinhibitors and μ-opioid receptor antagonists, are provided incombination in a synergistic antiproliferative and antimigratoryeffective amount. It will be understood, however, that the total dosageof the compounds and compositions of the invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically-effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex, and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, onetechnique is to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved.

If desired, an effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up a dailydose. As noted, those of ordinary skill in the art can readily optimizeeffective doses and co-administration regimens (as described herein) asdetermined by good medical practice and the clinical condition of theindividual patient.

Generally, oral doses of the μ-opioid receptor antagonists, particularlyperipheral antagonists, will range from about 0.01 to about 80 mg/kgbody weight per day. It is expected that oral doses in the range from 1to 20 mg/kg body weight will yield the desired results. Generally,parenteral administration, including intravenous and subcutaneousadministration, will range from about 0.001 to 5 mg/kg body weight. Itis expected that doses ranging from 0.05 to 0.5 mg/kg body weight willyield the desired results. Dosages may be adjusted appropriately toachieve desired drug levels, local or systemic, depending on the mode ofadministration. For example, it is expected that the dosage for oraladministration of the μ-opioid receptor antagonists in an entericallycoated formulation would be from 10 to 30% of the non-coated oral dose.In the event that the response in a patient is insufficient with suchdoses, even higher doses (or effectively higher than 30% dosage by adifferent, more localized delivery route) may be employed to the extentthat the patient tolerance permits. Multiple doses per day arecontemplated to achieve appropriate systemic levels of compounds.Appropriate system levels can be determined by, for example, measurementof the patient's plasma level of the drug using routine HPLC methodsknown to those skilled in the art.

In embodiments of the invention, an mTOR inhibitor compound may beadministered as appropriate, e.g. in dosages which are known forcompounds of embodiments of the invention, by any administration route,for example, enterally, (e.g., orally), or parenterally or topically. Avariety of oral and parental dosage forms are known for the mTORinhibitors. Oral daily dosages may range from 0.1 mg to 25 mg, in theform, e.g., of dispersible tablets. A weekly dosage may include up to 70mg, depending on the disease being treated. For parental administration,including intravenous administration, an initial intravenous dosage willbe between about 0.1 and 100 mg/m² when administered on a daily dosageregimen (daily for five days, every two to three weeks), and moresuitably, between 0.1 and 1000 mg/m² when administered on a once weeklydosage regimen. For example, everolimus may be administered orally, indaily dosages from 0.1 mg up to 25 mg or 0.1 mg to 15 mg, including 0.1mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 2.5 mg, 5 mg, or 10 mg, e.g., in theform of dispersible tablets or in the form of a solid dispersion,depending on the disease being treated. Everolimus may be administeredin a weekly dosage that may include up to 70 mg, such as 10 to 70 mg, or30 to 50 mg, depending on the disease being treated. For furtherexample, tacrolimus (Protopic) may be administered as an ointment of0.03% to 0.1% (w/w) in an ointment base. Other mTOR inhibitors may beadministered analogously, e.g. in similar dosage ranges.

In illustrated embodiments of the invention, the μ-opioid receptorantagonists are co-administered with an mTOR inhibitor. In other words,the co-administration of the μ-opioid receptor antagonist compound withan mTOR inhibitor, is suitably considered a pharmaceutical combinationwhich contains an μ-opioid receptor antagonist and an mTOR inhibitor,the combination being adapted for the administration of the peripheralμ-opioid receptor antagonist on a daily or intermittent basis, and theadministration of the mTOR inhibitor on a daily or intermittent basis.Thus, the μ-opioid receptor antagonists may be administered prior to,concomitant with, or after administration of the mTOR inhibitor. In anexemplary regimen, patients will receive a 30-minute intravenousinfusion of the mTOR inhibitor, followed immediately or preceded byadministration of the μ-opioid receptor antagonists. After one or moretreatment cycles, the dosages can be adjusted upwards or downwardsdepending on the results obtained and any side effects observed. In anillustrated embodiment, particularly suitable is administration of theμ-opioid receptor antagonist prior to administration of the mTORinhibitor.

Co-administrable agents in accordance with embodiments of the inventionalso may be formulated as an admixture, as, for example, in a singleformulation or single tablet. These formulations may be parenteral ororal, such as the formulations described, e.g., in U.S. Pat. Nos.6,277,384; 6,261,599; 5,958,452 and PCT Publication No. WO 98/25613,each hereby incorporated by reference.

Methods in accordance with embodiments of the invention can be usedalone or in conjunction with other treatments to control the growth ormigration of endothelial cells in connection with the various conditionsdescribed above. The mTOR inhibitor and the peripheral μ-opioid receptorantagonist may be co-administered with another therapeutic agent that isnot an opioid or μ-opioid receptor antagonist or an mTOR inhibitor. Suchsuitable therapeutic agents include other anticancer agents.

Embodiments of the invention also include the treatment of cancer. Thetypes of cancer that may be treated is limited only by the involvementof mTOR. Thus, it is contemplated that a wide variety of tumors may betreated using these therapies, including cancers of the brain, lung,liver, spleen, kidney, lymph node, pancreas, small intestine, bloodcells, colon, stomach, breast, endometrium, prostate, testicle, ovary,skin, head and neck, esophagus, bone marrow, blood or other tissue.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as “remission” and “reduction oftumor” burden also are contemplated given their normal usage.

Embodiments of the invention are further explained by the followingexamples, which should not be construed by way of limiting the scope ofthe present invention.

EXAMPLES Example 1 Inhibition of VEGF-Induced Akt Activation

To assess the effects of methylnaltrexone (MNTX) on the VEGF-inducedactivation of the serine/threonine kinase Akt, a well-characterizedendothelial cell line, human pulmonary microvascular endothelial cells(HPMVEC), was used. HPMVEC were serum starved for one hour and eitheruntreated (control) or treated with VEGF (100 nM, 5 minutes) with orwithout pre-treatment (1 hour) with 100 nM MNTX, 100 ng/ml bevacizumab,100 μM 5-FU, 100 nM MNTX+100 ng/ml bevacizumab or 100 nM MNTX+100 μM5-FU. Cell lysates were obtained, run on SDS-PAGE and immunoblotted withanti-pSer⁴⁷³Akt, anti-pThr³⁰⁸Akt, anti-Akt or anti-actin antibody. Theresults indicated that methylnaltrexone abrogated VEGF-inducedphosphorylation of Akt at the serine and threonine at positions 473 and308, respectively (FIG. 2). Furthermore, methylnaltrexone in combinationwith bevacizumab and 5-FU synergistically inhibited Akt activation. Theeffects of various concentrations of MNTX (1.0, 10, 100, 1000 nM) oninhibition of VEGF-induced pSer⁴⁷³Akt (FIG. 3A) and pThr³⁰⁸Akt (FIG. 3B)immunoreactivity demonstrated that this inhibition is dose-dependent.

Cell Culture and Reagents—

Human pulmonary microvascular EC were obtained from Cambrex(Walkersville, Md.) and cultured as previously described (Singleton etal. (2006), Microvasc. Res. 72(1-2):3-11; Singleton et al. (2007), Am.J. Respir. Cell Mol. Biol. 37(2):222-231) in EBM-2 complete medium(Cambrex) at 37° C. in a humidified atmosphere of 5% CO₂, 95% air, withpassages 6-10 used for experimentation. Unless otherwise specified,reagents were obtained from Sigma (St. Louis, Mo.). Vascular endothelialgrowth factor (VEGF) was purchased from R&D Systems (Minneapolis,Minn.). Methylnaltrexone bromide (MNTX) was purchased from MallinckrodtSpecialty Chemicals (Phillipsburg, N.J.). Bevacizumab was purchased fromGenentech (South San Francisco, Calif.). 5-fluorouracil (5-FU) waspurchased from Abraxis Pharmaceutical Products (Schaumburg, Ill.).Naltrexone and rapamycin were purchased from Sigma (St. Louis, Mo.).Reagents for SDS-PAGE electrophoresis were purchased from Bio-Rad(Richmond, Calif.) and Immobilon-P transfer membrane was purchased fromMillipore (Millipore Corp., Bedford, Mass.). Rabbit anti-pSer⁴⁷³Akt,rabbit anti-pThr³⁰⁸Akt and rabbit anti-Akt antibodies were purchasedfrom Cell Signaling Technologies (Danvers, Mass.). Mouse anti-β-actinantibody was purchased from Sigma (St. Louis, Mo.). Secondaryhorseradish peroxidase (HRP)-labeled antibodies were purchased fromAmersham Biosciences (Piscataway, N.J.).

SDS-PAGE and Immunoblotting—

Cellular materials from treated or untreated HPMVEC were incubated withIP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl₂, 1% NonidetP-40 (NP-40), 0.4 mM Na₃VO₄, 40 mM NaF, 50 μM okadaic acid, 0.2 mMphenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem proteaseinhibitor mixture 3), subjected to SDS-PAGE in 4-15% polyacrylamidegels, transferred onto Immobilon™ membranes, and developed with specificprimary and secondary antibodies. Visualization of immunoreactive bandswas achieved using enhanced chemiluminescence (Amersham Biosciences). Inorder to investigate the relative amount of activated Akt, thepSer⁴⁷³Akt and pThr³⁰⁸Akt immunoreactive band intensities were dividedby total Akt immunoreactive band intensity.

Example 2 Effect of Rapamycin on VEGF-Induced Endothelial Cell Migrationand Proliferation

To investigate the role of mTOR in cellular migration and proliferation,the effect of rapamycin on HPMVEC migration and proliferation assays wasdetermined. Human EC were assayed for VEGF (100 nM)-inducedprofileration (FIG. 4) and migration (FIG. 5) in the presence or absenceof 0.01, 0.1, 1.0 or 10 nM rapamycin. The results demonstrate thatinhibition of mTOR by rapamycin results in a dose-dependent inhibitionof endothelial cell proliferation and migration. This is in agreementwith previous studies demonstrating that mTOR is involved in theseprocesses.

Human Pulmonary Microvascular EC Migration Assay—

Twenty-four transwell units with 8 μM pore size were used for monitoringin vitro cell migration. HPMVEC (˜1×10⁴ cells/well) were plated withvarious treatments to the upper chamber and VEGF (100 nM) was added tothe lower chamber. Cells were allowed to migrate for 18 hours. Cellsfrom the upper and lower chamber were quantitated using the CellTiter96™MTS assay (Promega, San Luis Obispo, Calif.) and read at 492 nm. Percentmigration was defined as the # of cells in the lower chamber divided bythe number of cells in both the upper and lower chamber. Each assay wasset up in triplicate, repeated at least five times and analyzedstatistically by Student's t test (with statistical significance set atP<0.05).

Human Pulmonary Microvascular EC Proliferation Assay—

For measuring cell growth, HPMVEC [5×10³ cells/well] pretreated withvarious agents were incubated with 0.2 ml of serum-free media containing100 nM VEGF for 24 h at 37° C. in 5% CO₂195% air in 96-well cultureplates. The in vitro cell proliferation assay was analyzed by measuringincreases in cell number using the CellTiter96™ MTS assay (Promega, SanLuis Obispo, Calif.) and read at 492 nm. Each assay was set up intriplicate, repeated at least five times and analyzed statistically byStudent's t test (with statistical significance set at P<0.05).

Statistical Analysis—

Student's t test was used to compare the means of data from two or moredifferent experimental groups. Results are expressed as means±S.E.

Example 3 Synergistic Effect of Methylnaltrexone and mTOR Inhibitors onVEGF-Induced Endothelial Cell Migration and Proliferation

Given the complex feedback inhibition signaling network that existsbetween Akt and mTOR, the migration and proliferation assays describedabove were used to determine whether the simultaneous inhibition of mTORactivation using rapamycin and inhibition of Akt using methylnaltrexoneproduced a synergistic effect on these processes. Human endothelialcells were assayed for VEGF (100 nM)-induced profileration (FIG. 6) andmigration (FIG. 7) in the presence or absence of 10 or 100 nM MNTX, 10nM naltrexone, 0.1 nM rapamycin, 100 nM MNTX+0.1 nM rapamycin or 10 nMnaltrexone+0.1 nM rapamycin. Similar results were obtained withmethylnaltrexone in combination with temsirolimus (FIG. 10).

The results demonstrate that methylnaltrexone and mTOR inhibitorssynergistically inhibit the migration and proliferation of endothelialcells. Unlike the results observed for methylnaltrexone, treatment ofhuman endothelial cells with temsirolimus did not inhibit VEGF-inducedactivation of Akt (FIG. 8). This indicates that the observed synergisticeffect of methylnaltrexone in combination with mTOR inhibitors is likelydue to inhibition of the mTOR signaling pathway at two distinct points,with methylnaltrexone-induced inhibition occurring upstream of Aktactivation and inhibition by mTOR inhibitors occurring downstream of Aktactivation (FIG. 12). This hypothesis is bolstered by the fact thatmethylnaltrexone inhibits VEGF-induced formation of mTOR Complex I andmTOR Complex II, while temsirolimus inhibits the formation of mTORcomplex II only (FIG. 9A).

To further elucidate the mechanism of VEGF-induced Akt activation, humanendothelial cells treated with VEGF with or without pretreatment withthe PI3 kinase inhibitor, LY294002, Src siRNA or Rictor (mTOR complex IIcomponent) siRNA. Lysates were immunoblotted with anti-pSer⁴⁷³Akt,anti-pThr³⁰⁸Akt or anti-Akt antibody (FIG. 9B). PI3 kinase inhibitorinhibits VEGF-induced Akt threonine 308 phosphorylation. Inhibiting mTORComplex 2 formation (Rictor siRNA) blocks VEGF-induced Akt serine 473phosphorylation. Inhibiting Src expression (siRNA) blocks both Aktserine 473 and Akt threonine 308 phosphorylation. Further, the centralrole of tyrosine phosphatase (PTP) in the synergistic effect ofmethylnaltrexone in combination with mTOR inhibitors on humanendothelial cell proliferation and migration was demonstrated using thepotent PTP inhibitor 3,4-dephostatin, which inhibited these VEGF-inducedpro-angiogenic events (FIG. 11).

MNTX and Temsirolimus Regulation of Akt Phosphorylation—

Human EC were serum starved for one hour and either untreated (control)or treated with VEGF (100 nM, 5 minutes) with or without pretreatment (1hour) with 100 nM MNTX or 100 nM temsirolimus. EC lysates were obtained,run on SDS-PAGE and immunoblotted with anti-pSer⁴⁷³Akt, anti-pThr³⁰⁸Aktor anti-Akt antibody.

Analysis of mTOR Complex Formation and Regulation of AktPhosphorylation—

Human EC were serum starved for one hour and either untreated (control)or treated with VEGF (100 nM, 5 minutes) with or without pretreatment (1hour) with 100 nM MNTX or 100 nM Temsirolimus. EC lysates were obtainedand immunoprecipitated with anti-Raptor (mTOR Complex 1 component) oranti-Rictor (mTOR complex 2 component) antibody. The immunoprecipitatedmaterial was run on SDS-PAGE and immunoblotted with either anti-mTOR,anti-FKBP12, anti-Raptor, anti-SIN1 or anti-Rictor antibody. Toinvestigate the regulation of Akt phosphorylation, human EC were serumstarved for one hour and either untreated (control) or treated with VEGF(100 nM, 5 minutes) with or without pretreatment with the PI3 kinaseinhibitor, LY294002 (10 μM, 1 hour), Src siRNA or Rictor (mTOR complex 2component) siRNA. EC lysates were obtained, run on SDS-PAGE andimmunoblotted with anti-pSer⁴⁷³Akt, anti-pThr³⁰⁸Akt or anti-Aktantibody.

Synergistic Effects of MNTX with Temsirolimus on Inhibition ofVEGF-Induced Human EC Proliferation and Migration.

Inhibition curves of human EC assayed for VEGF (100 nM)-inducedproliferation and migration (24 hours) in the presence or absence of0.1, 1.0, 10, 100 or 500 nM MNTX, Temsirolimus or 10 nMMNTX+Temsirolimus. MNTX inhibited EC VEGF-induced proliferation with anIC50 of ˜100 nM. Adding 10 nM MNTX to EC shifted the IC50 ofTemsirolimus inhibition of VEGF-induced proliferation from ˜10 nM to ˜1nM. Experiments were performed in triplicate. Error bars=standarddeviation.

MNTX Synergy with Temsirolimus is Regulated by Tyrosine PhosphataseActivity.

Human EC were assayed for VEGF (100 nM)-induced proliferation andmigration (24 hours) in the presence of 10 nM or 15 nM Temsirolimus(IC50 concentrations for inhibition of proliferation in the absence orpresence of 3,4-Dephostatin, respectively) with or without 10 nM MNTX.Experiments were performed in triplicate. Error bars=standard deviation.

While not wishing to be limited by any particular theory, FIG. 12depicts a possible mechanism for the synergy demonstrated by the resultsof the Examples.

Example 4 Treatment of Mammalian Subjects with mTOR Inhibitors inCombination with Methylnaltrexone

In a first set of experiments, mice are induced to develop tumors bytransformation, inbreeding or transplantation of tumor cells.Forty-eight mice, each bearing tumors having a volume of at least 60mm³, are randomly divided into four groups. The first group receives acontrol substance comprising neither an opioid antagonist nor an mTORinhibitor. The second group receives the peripheral opioid antagonistmethylnaltrexone administered via an acceptable route to contact thetumor with a therapeutically effective amount of methylnaltrexone, e.g.,oral administration at a dose of 5 mg/kg/day. The third group receivesthe mTOR inhibitor, such as rapamycin, administered via an acceptableroute to contact the tumor with a therapeutically effective amount ofmTOR inhibitor, e.g., injection of rapamycin at a dose of 1 mg/kg/day.The forth group receives a combination of methylnaltrexone and mTORinhibitor.

Differences in the rate of tumor growth, tumor size, angiogenesis withinthe tumor and mortality between each group of mice are recorded.Additional experiments will be performed using varied treatment doses ofmTOR inhibitors, such as rapamycin, to determine the reduction intherapeutic dose of the mTOR inhibitor resulting from co-administrationwith methylnaltrexone.

Example 5 Treatment of Mammalian Subjects with mTOR Inhibitors inCombination with Alvimopan

In a first set of experiments, mice are induced to develop tumors bytransformation, inbreeding or transplantation of tumor cells.Forty-eight mice, each bearing tumors having a volume of at least 60mm³, are randomly divided into four groups. The first group receives acontrol substance comprising neither an opioid antagonist nor an mTORinhibitor. The second group receives the peripheral opioid antagonistalvimopan administered via an acceptable route to contact the tumor witha therapeutically effective amount of alvimopan. The third groupreceives the mTOR inhibitor, such as rapamycin, administered via anacceptable route to contact the tumor with a therapeutically effectiveamount of mTOR inhibitor, e.g., injection at a dose of 1 mg/kg/day. Theforth group receives a combination of alvimopan and mTOR inhibitor.

Differences in the rate of tumor growth, tumor size, angiogenesis withinthe tumor and mortality between each group of mice are recorded.Additional experiments will be performed using varied treatment doses ofmTOR inhibitors, such as rapamycin, to determine the reduction intherapeutic dose of the mTOR inhibitor resulting from co-administrationwith alvimopan.

In summary, methods in accordance with embodiments of the invention areprovided for treating a disease or disorder associated withproliferation and migration of cells, including cancer and otherhyperproliferative diseases as well as autoimmune disease, which methodsinclude co-administration of an mTOR inhibitor and a μ-opioid receptorantagonist.

The invention has now been described with reference to various specificembodiments and techniques. However, it should be understood that manyvariations and modifications may be made while remaining within thespirit and scope of the invention.

All publications, patents, and patent applications are herein expresslyincorporated by reference to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated by reference. In case of conflict between thepresent disclosure and the incorporated patents, publications andreferences, the present disclosure should control.

1. A method of treating a disorder characterized by abnormal migrationand/or proliferation of cells, the method comprising administering to asubject in need thereof a synergistically effective amount of a mTORinhibitor and a mu-opioid receptor antagonist
 2. The method of claim 1,wherein the mTOR inhibitor is TOP216, OSI-027 or a rapamycin derivative,which is temsirolimus, everolimus, deforolimus, TAFA93, AP23573, ABT-573or nab-rapamycin.
 3. The method of claim 1, wherein the mu-opioidreceptor antagonist is a peripheral mu-opioid receptor antagonist. 4.The method of claim 2, wherein the mu-opioid receptor antagonist is aperipheral mu-opioid receptor antagonist.
 5. The method of claim 1,wherein the mu-opioid receptor antagonist is not a quaternary derivativeof noroxymorphone.
 6. The method of claim 2, wherein the mu-opioidreceptor antagonist is not a quaternary derivative of noroxymorphone. 7.The method of claim 5, wherein the mu-opioid receptor antagonist ispegylated naloxone or alvimopan.
 8. The method of claim 6, wherein themu-opioid receptor antagonist is pegylated naloxone or alvimopan.
 9. Themethod of claim 3, wherein the peripheral mu-opioid receptor antagonistis a N-substituted piperidines.
 10. The method of claim 4, wherein theperipheral mu-opioid receptor antagonist is a N-substituted piperidines.11. The method of claim 9, wherein the N-piperidine is apiperidine-N-alkylcarbonylate.
 12. The method of claim 10, wherein theN-piperidine is a piperidine-N-alkylcarbonylate.
 13. The method of claim11, wherein the the piperidine-N-alkylcarbonylate is aN-alkylamino-3,4,4 substituted piperidine.
 14. The method of claim 12,wherein the the piperidine-N-alkylcarbonylate is a N-alkylamino-3,4,4substituted piperidine.
 15. The method of claim 13, whereinN-alkylamino-3,4,4 substituted piperidine is alvimopan.
 16. The methodof claim 14, wherein N-alkylamino-3,4,4 substituted piperidine isalvimopan.
 17. The method of claim 1, wherein the mu-opioid antagonistis a tertiary ammonium derivative of morphinan, benzomorphan andnormorphinan.
 18. The method of claim 2, wherein the mu-opioidantagonist is a tertiary ammonium derivative of morphinan, benzomorphanand normorphinan.
 19. The method of claim 1, wherein the mu-opioidantagonist is polymeric conjugate of a mu-opioid antagonist.
 20. Themethod of claim 2, wherein the mu-opioid antagonist is polymericconjugate of a mu-opioid antagonist.
 21. The method of claim 19, whereinthe polymeric conjugate is a pegylated naloxone or naltrexone.
 22. Themethod of claim 21, wherein the the polymeric conjugate is pegylatednaloxone.
 23. The method of claim 1, wherein the cells are endothelialcells.
 24. The method of claim 23, wherein the endothelial cells arevascular endothelial cells and the unwanted migration and/orproliferation of the vascular endothelial cells is abnormal/unwantedangiogenesis.
 25. The method of claim 1, wherein the disorder is acancer, diabetes, sickle cell anemia, a vascular wound, or aproliferative retinopathy.
 26. The method of claim 25, wherein thecancer is cancer of the brain, lung, liver, stomach, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,bone marrow or blood.
 27. The method of claim 1, wherein the mu-opioidreceptor antagonist and mTOR inhibitor are administered at the sametime, the mu-opioid receptor antagonist is administered prior toadministering the mTOR inhibitor, or the mu-opioid receptor antagonistis administered after administering the mTOR inhibitor.
 28. The methodof claim 1, wherein the administering of the mu-opioid receptorantagonist, the mTOR inhibitor, or both is oral, sublingual,intramuscular, subcutaneous, intravenous, topical or transdermal.
 29. Amethod of inhibiting growth factor signaling in mammalian cells,comprising contacting the cells with a synergistically effective amountof an mTOR inhibitor and a mu-opioid receptor antagonist.
 30. The methodof claim 29, wherein the growth factor signaling is VEGF signaling. 31.A method of achieving an effect in a subject comprising administering tothe subject a synergistically effective amount of the mTOR inhibitor andthe mu-opioid receptor antagonist, wherein the effect is inhibitingcellular hyperproliferation, treating cancer, inhibiting tumor growth,improving the therapeutic utility of an mTOR inhibitor, reducing adverseside effects associated with treatment with an mTOR inhibitor, ortreating an autoimmune disease.
 32. The method of claim 31, wherein theautoimmune disease is allergic encephalomyelitis, insulin-dependentdiabetes mellitus, lupus, rheumatoid arthritis, multiple sclerosis,dermatomyositis, Grave's disease or adjuvant arthritis.
 33. Apharmaceutical combination comprising an mTOR inhibitor and a mu-opioidreceptor antagonist in synergistically effective amounts.
 34. Thepharmaceutical combination of claim 33, wherein the mu-opioid antagonistis a pegylated naloxone or alvimopin.
 35. The method of claim 1, whereinthe mu-opioid antagonist is a piperidine-N-alkylcarboxylate representedby formula (III):

wherein R¹ is hydrogen or alkyl; R² is hydrogen, alkyl, or alkenyl; R³is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl; R⁴ is hydrogen, alkyl, or alkenyl; A is OR⁵ orNR⁶R⁷; wherein R⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, oraryl-substituted alkyl; R⁶ is hydrogen or alkyl; R⁷ is hydrogen, alkyl,alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,cycloalkenyl-substituted alkyl or aryl-substituted alkyl, oralkylene-substituted B or together with the nitrogen atom to which theyare attached, R⁶ and R⁷ form a heterocyclic ring selected from pyrroleand piperidine; B is

wherein R⁵ is hydrogen or alkyl; R⁹ is hydrogen, alkyl, alkenyl, aryl,cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,cycloalkenyl-substituted alkyl or aryl-substituted alkyl or togetherwith the nitrogen atom to which they are attached, R⁵ and R⁹ form aheterocyclic ring selected from pyrrole and piperidine; W is OR¹⁰,NR¹¹R¹², or OE; wherein R¹⁰ is hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substitutedalkenyl, or aryl-substituted alkyl; R¹¹ is hydrogen or alkyl; R¹² ishydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl,aryl-substituted alkyl, or alkylene-substituted C(═O)Y or, together withthe nitrogen atom to which they are attached, R¹¹ and R¹² form aheterocyclic ring selected from pyrrole and piperidine; E is

alkylene-substituted (C═O)D, or —R¹³OC(═O)R¹⁴; wherein R¹³ isalkyl-substituted alkylene; R¹⁴ is alkyl; D is OR¹⁵ or NR¹⁶R¹⁷; whereinR¹⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,cycloalkyl-substituted alkyl, cycloalkenyl substituted alkyl, oraryl-substituted alkyl; R¹⁶ is hydrogen, alkyl, alkenyl, aryl,aryl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkyl substitutedalkyl, or cycloalkenyl-substituted alkyl; R¹⁷ is hydrogen or alkyl or,together with the nitrogen atom to which they are attached, R¹⁶ and R¹⁷form a heterocyclic ring selected from the group consisting of pyrroleor piperidine; Y is OR¹⁸ or NR¹⁹R²⁰; wherein R¹⁸ is hydrogen, alkyl,alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R¹⁹ ishydrogen or alkyl; R²⁰ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl,cycloalkenyl, cycloalkyl substituted alkyl, cycloalkenyl-substitutedalkyl, or aryl-substituted alkyl or, together with the nitrogen atom towhich they are attached, R¹⁹ and R²⁰ form a heterocyclic ring selectedfrom pyrrole and piperidine; R²¹ is hydrogen or alkyl; and n is 0 to 4.36. The method of claim 35, wherein the piperidine-N-alkylcarbonylate isa N-alkylamino-3,4,4 substituted piperidine represented by formula (IV):


37. A method of treating a disorder characterized by abnormal migrationand/or proliferation of cells, the method comprising administering to asubject in need thereof an effective amount of a mTOR inhibitor and amu-opioid receptor antagonist.